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Chapter 3

HPLC Method development and validation of protein based drugs

67

CHAPTER 3

HPLC METHOD DEVELOPMENT

AND VALIDATION

OF PROTEIN BASED DRUGS

Chapter 3


68

3.1 INTRODUCTION

The advent of recombinant DNA technology has led to a worldwide zeal to develop

protein pharmaceuticals in the past three decades. These protein pharmaceuticals

include functional regulators and supplements, enzyme activators and inhibitors, poly-

and monoclonal antibodies, and various vaccines. In comparison with small chemical

drugs, protein pharmaceuticals have high specificity and activity at relatively low

concentrations. These features have made protein pharmaceuticals indispensable in

combating human diseases.

Due to advances in analytical separation technology, recombinant proteins can now be

purified to an unprecedented level (Bond et al., 1998). Highly purified protein

pharmaceuticals significantly reduce the known and unknown potential side or even

toxic effects. [1]

However, one of the most challenging tasks remaining in the development of protein

pharmaceuticals is dealing with physical and chemical instabilities of proteins. Protein

instability is one of the two major reasons why protein pharmaceuticals are

administered traditionally through injection rather than taken orally like most small

chemical drugs (Wang, 1996). Protein pharmaceuticals usually have to be stored

under cold conditions or even freeze-dried to a solid form to achieve an acceptable

shelf life. [2]

Pharmaceutical excipients may be added to a formulation to stabilize the protein, to

aid in manufacture of the dosage form, for control or target delivery in the body, or

provide tonicity to minimize pain upon injection. Examples include buffers,

carbohydrates as bulking agents for lyophilization, polymers as viscosity agents for

topical applications, and salts or sugars for adjusting solution osmolality into a

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physiological range. Although it is often assumed that pharmaceutical excipients are

essentially inert, some additives may have certain toxicological or biological

activities, and therefore, may play a role in defining the overall safety profile of a

drug. Although excipients are selected for their low toxicity, and are generally well

tolerated, certain excipient classes such as antioxidants and preservatives may have

some level of toxicity associated with them [3-5]. The overall safety profile of a drug

or excipient is not determined independently from each other, since the combination

of drug and excipient together defines the drug product tested in clinical trials [6].

Many pharmaceutical excipients are classified as “generally regarded as safe” or

GRAS and typically have a long history of safe use as food additives [7].

One major hurdle for the formulation scientist to develop stable protein formulations

is the limited number of excipients commonly used in parenteral formulations. The

introduction of novel pharmaceutical excipients to stabilize proteins would be of great

interest, however, the safety and efficacy of these new compounds would need to be

evaluated as part of the drug approval process [6]. Novel pharmaceutical excipients

have been designed to enhance protein stability, for example, low molecular weight

multi-ions [8].

The various classes of pharmaceutical excipients commonly used to formulate and

stabilize protein therapeutic drugs and vaccines are shown in Table 3.1,.(science

protein excipients)

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Table 3.1 Pharmaceutical excipients for use in protein formulations (categories,

examples, comments

Category Representative examples General comments Cautionary comments

Buffering agents

Amino acids

Osmolytes

Sugars and

cargohydrates

Protein and

polymers

Salts

Surfactants

Chelators and

anti-oxidants

Preservatives

Specific ligands

Citrate acetate, histidine, phosphate, Tris

Histidine, arginine, glycine, proline,

lysine, methionline

Sucrose, trehalose, sorbitol, glycine,

proline, glutamate, glyceron, ures

Sucrose, trehalose, sorbitol, mannitol,

glucose, lactose

HSA, gelatin, PVP, PLGA, PEG

Sodium chloride, Potassium chloride,

Sodium sulfate

Polysorbate 20 and 80

EDTA, DTPA, amino acids (His, Met),

ethanol

Benzyl alcohol, m-cresol, phenol

Metals, ligands, amino acids, polyanions

Maintain solution pH

Buffer-ion specific interactions with protein

Specific interactions with protein

Antioxidant (His, Met)

Buffering and tonicifying agents

Natural compounds that stabilize proteins and

macromolecules against environmental stress

(temperature, dehydration

Protein stablilizer in liquid and lyophilized

states

Tonicifying agents

Lactose as a carrier for inhaled drugs

Dextrose solutions during IV administration

Competitive inhibitor of protein adsorption

Lyophilization bulking agents

Drug delivery vehicles

Tonicifying agents

Stabilizing or destabilizing effects on proteins,

especially with anions (Hofmeister salt series)

Competitive inhibitor of protein adsorption

Competitive inhibitor of protein surface

denaturation

Liposomes as drug delivery vehicles

Bind metal ions

Free radical scavengers

Prevents microbial growth in multi-dose

formulations

Binds protein and stabilizes native

conformation against stress induced unfolding

Binding may also affect protein’s

conformational flexibility

pH may change with temperature

Crystallizaiton during freezing.

Decomposition during storage

High concentration often required for

stabilization

Many additional osmolytes have been identified,

but not currently approved for use as

pharmaceutical excipients

Destabilizing effects also reported

Reducing sugars react with proteins to form

glycated proteins

Nonreducing sugars can hydrolyze forming

reducing sugars

Impurities such as metals and 5-HMF

Trends toward use of recombinant sources of

HSA and gelatins

Drug delivery polymers may not be compatible

with protein drugs.

Concentration dependent effects

Trace metals can cause oxidation

May be corrosive to metal surfaces

Lowers Tg’ of solution (may affect

lyophilization)

Peroxide can cause oxidation

May degrade during storage

Complex behaviour during membrane filtration

due to micelle formation

Certains antioxidant such as ascorbic acid and

glutathione lead to protein stability

Light exposure accelerates oxidation

Inverse concentration dependant effects on

protein destabilization vs antimicrobial

effectiveness

May involve use of novel excipients or an

excipient with biological activity

The higher complexity of peptides and proteins compared to organic low molecular

weight drug substances and the different ways to produce biotechnological products

lead to special requirements concerning their quality assurance and analytical testing.

Erythropoietin (EPO), the major physiological regulator of the red blood cell

formation, is produced primarily by the kidneys and excreted in the urine. Production

of EPO is stimulated under conditions of hypoxia and it exerts its biological effect by

binding to specific receptors on erythroid progenitor cells in the bone marrow. Human

EPO has an apparent molecular weight of 30,000 Da, consists of 165 amino acids and

contains two disulfide linkages.

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The first clinical trials were performed with human erythropoietin purified from urine.

Today it is possible to produce human EPO by recombinant DNA technology (Lin et

al.,1985) [9] (rHuEP0) in mammalian cell cultures receiving much better yields to

supply the pharmaceutical market since its introduction in 1988. Recombinant human

erythropoietin is therapeutically used for the treatment of anemia resulting from

chronic kidney failure or from cancer therapy. Clinical trials with recombinant human

erythropoietin showed its efficacy for reversing anemia related to advanced cancer or

chemotherapy of cancer (Caro et al.,1989) [10]

Proteins or peptides can also be produced in quantities sufficient for pharmaceutical

use by recombinant DNA technology in bacteria, yeasts, or in cell cultures (Nagata et

al., 1980; Murray, 1980). [11, 12] The gene of interest is cloned into the appropriate

host where the recombinant protein is then expressed. After synthesis of the

recombinant protein by the ribosomes of the host cell, it is either directly secreted or

has to be isolated after disruption of the host cell. Depending on the host cell also

post-translational modifications of the recombinant protein occur. The recombinant

protein is then purified to homogeneity in several steps. The pure substance is

characterized and tested for the absence of impurities like host cell proteins or

aggregates. Somatostatin was the first human hormone synthesized in cell cultures.

Nowadays it is possible to produce human erythropoietin (HuEPO) by recombinant

DNA technology (Lin et al.,1985) [9] in mammalian cell cultures [e.g., Chinese

hamster ovary (CHO) cells]. Much higher yields compared to the purification from

urine are of great advantage due to the steadily increasing demand for the product

since its introduction to the market in 1988. Hu EPO consists of a polypeptide

sequence of 165 amino acids and a carbohydrate moiety, which contributes about

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40% of the molecular weight and is attached at four glycosylation sites. Asn24, Asn38

and Asn83 are N-glycosylated whereas Ser126 was found to be O-glycosylated (Lai et

al., 1986). [13] ( science erythro)

Figure 3.1 Structure of EPO

The biological activity of EPO in vivo is affected by the glycosylation pattern (sialic

acid content). Since production system and process conditions for rhEPO affect the

glycosylation pattern the production process should be carefully validated and

monitored to assure consistency of the biological activity throughout different

production batches [14].

At present the content of rhEPO preparations is typically tested by complex in vivo

potency assays which measure the relevant biological activity. For instance, the

European Pharmacopoeia describes an assay for rhEPO bulk solutions in which the

effect of rhEPO on mice kept under low oxygen conditions is monitored by measuring

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incorporation of radio-labelled ferric chloride [15]. For assaying the content of rhEPO

preparations in a routine setting these types of bioassays require a significant number

of animals. A rapid and less resource demanding physico-chemical assay may not

specifically mimic bioactivity but it would provide a wider forum for controlling the

quality of these common pharmaceutical products. Moreover, from an analytical point

of view content assays based on physicochemical technology will be more precise

than bioassays. Developing a suitable physicochemical assay for rhEPO preparations

is hampered by the low dose of the micro-heterogeneous glycoprotein in presence of

relatively large amounts of excipients. Particular difficulties are encountered when

human serum albumin (HSA) is present. The protein HSA is obtained from large

pools of human plasma and cannot be considered chemically homogeneous. The

physicochemical assays should have a high degree of selectivity and reproducibility

for rhEPO assay. So far capillary electrophoresis (CE) methods have been developed

to characterize the rhEPO glycoform pattern and a capillary zone electrophoresis

method has been prescribed by the European Pharmacopoeia as an identification test

for rhEPO in concentrated bulk solutions [15]. In addition to this method, another CE

method has been developed that is capable of analysing rhEPO pharmaceutical

preparations containing salts and HSA, and in the concentration range of 0.03–1.92

mg rhEPO/ml [16].

High-performance anion-exchange chromatography (HPAEC) separates proteins

according to their negative electric charge and has been used for EPO assay with

fluorimetric detection by D.M.A.M. Luykx et al. in pharmaceutical products. [17].

Srinivas R. Gunturi et al. have developed a method for the determination of rHu EPO

aggregates in formulations by HPLC method with fluorescence detection [18].

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Among the possible methods to eliminate HSA, immunoaffinity chromatography

(IAC) is one of the most effective ones [19]. However, there is no method reported

for the determination of EPO in the presence of HSA without any sample

pretreatment.

HPLC in combination with UV-detection is a separation method that provides a

powerful means for characterising the homogeneity of common biopharmaceuticals

such as somatropin, insulin and interferons. Because of its high resolution, reversed-

phase HPLC is often applied for quantification of the active pharmaceutical ingredient

and for the analysis of closely related protein variants or degradation products (e.g.,

oxidised, deamidated) [20-22].

Although HPLC methodologies have been described previously, they have been

developed either for analysis of purified r-Hu EPO monomeric protein [23-24] or for

investigation of r-Hu EPO (monomer) metabolic pathways [25] in the absence of

HSA.

3.2 CHALLENGES IN RP- HPLC / UPLC ANALYSIS OF PROTEINS AND

PEPTIDES

RP-HPLC / UPLC analysis of biomolecules such as proteins and peptides can be a

challenge as there are often problems associated with analytical systems such as

excessive band broadening, peak tailing or misshaped bands, low recovery, ghost

peaks and the appearance of one protein in two or more distinct bands [26].

Understanding the impact of process variables in RP-HPLC can help minimise or

eliminate these undesirable effects.

The analysis of biochemical entities such as peptides, proteins, and oligo-nucleotides

by RP-HPLC pose different challenges as compared to the analysis of small chemical

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molecules since they have larger hydrodynamic radii and different functionalities in

the molecules that may result in different interactions with RP-HPLC stationary and

mobile phases. These factors must be considered in the development of an analytical

method for proteins and peptides [26-27]

However, there is no method reported for the determination of EPO in the presence of

HSA without any sample pretreatment

The objective of the study was to develop methods, using “RP-HPLC, and UPLC”

techniques that enable quantification of EPO in medicinal formulations containing

HSA.

Abbreviations used:

EPO – Erythropoietin

EPO-IRS – EPO Internal Reference Standard

DS (API) – Drug Substance (Active Pharmaceutical Ingredient)

EPO-DS – EPO Drug Substance

DP – Drug Product

EPO-DP – EPO Drug Product

RMP – Reference

rHu – Recombinant Human

HSA – Human Serum Albumin

EP – European Pharmacopoeia

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3.3 QUANTIFICATION OF EPO AND METHOD VALIDATION

3.3.1 EXPERIMENTAL

Materials, reagents and chemicals

HPLC grade acetonitrile was purchased from Merck; tri-fluoro-acetic acid (TFA) was

purchased from Sigma Aldrich. Ultra pure water was obtained using Milli-Q® UF-

Plus (Millipore) system. Human Serum Albumin (HSA) with 20% globulin fraction

was obtained from Baxter. EPO internal reference standard (EPO-IRS) having

0.8mg/mL concentration was procured from Intas Biopharmaceuticals Ahmedabad

was used as standard and was qualified using EP reference standard. Formulated EPO

(Drug Product) was used to prepare samples. Other chemicals, such as tri-sodium

dihydrate, sodium chloride and citric acid used were of “highest purity” available.

Preparation of standard, mobile phase and dilution buffer

EPO-IRS was used for preparation of different working standards using dilution

buffer or dilution buffer containing 2.5mg/mL of HSA.

Mobile phase ‘A’ consisted of 0.1% v/v TFA in Milli Q water and mobile phase ‘B’

consisted of 0.1% v/v TFA in acetonitrile.

Dilution buffer (Citrate buffer) containing 5.8 mg/mL tri-sodium dihydrate; 5.8

mg/mL sodium chloride and 0.06 mg/mL citric acid in “Milli Q water” was prepared

and used so as to have a matrix similar to EPO formulation. Dilution buffer with

HSA was prepared by diluting 2.5 mg/mL of HSA in dilution buffer. All dilutions

were made using calibrated digital micro-pipettes.

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Chromatographic condition

HPLC – An LC system equipped with an injection valve (quaternary), 215 UV

detector and chemstation software was used for RP-HPLC method. A reverse -phase

C8 column (4.6 mm ID × 250 mm L, porosity 300º A, particle size 5 µm) with guard

column (reverse-phase C18 column of 4.6 mm ID × 35 mm L, porosity 300º A,

particle size 5 µm) was used for separation. To get the optimum results, mobile phase

with a flow rate of 1.5 mL/min and column temperature at 45oC were used. The

gradient programme for mobile phase was optimized using a timed gradient

programme T (min)/%mobile phase A: 0/65, 4/65, 12/50, 14/50, 15/40, 16/65, 20/65.

UPLC – An LC system equipped with an injection valve (binary), a 210 UV detector

and Empower software was used for RP-UPLC method. Reverse-phase C18 column

(2.1 mm ID × 50 mm L, porosity 135ºA, particle size 1.7 µm) was used for

separation. To get the optimum results, mobile phase flow rate was kept constant at

0.35 mL/min and column temperature at 60oC. The gradient programme for mobile

phase was optimized using a timed gradient programme T (min)/%mobile phase A:

0/85, 0.12/85, 0.33/70, 0.62/64, 2.62/35, 3.19/0, 3.76/85 and 4.05/85.

3.3.2. RESULTS AND DISCUSSION

3.3.2.1. RP HPLC method

3.3.2.1.1 Method development

Initially, the gradient HPLC conditions were optimized for EPO in presence of HSA.

Based on the different hydrophobic properties of both proteins in a non-polar

stationary phase, an RP-HPLC in gradient mode was used. JADWIGA et. al. had

reported a HPLC method with an analysis time of about 60 min., with retention times,

of approximately 17 and 33 min for HSA and EPO, respectively The HPLC method

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proposed by JADWIGA et al. [28] was taken into consideration for the experiments

and efforts were made to minimize the analysis time which is a must for multi-product

facility. The chromatographic separation was achieved by applying chromatographic

conditions as described in above section 3.4.1.

The applied chromatographic conditions permitted a good separation of HSA and

EPO at different concentrations of EPO each containing 2.5 mg/mL of HSA. No

interference of HSA and other excipients was observed during the analysis as shown

in Figure 3.2.

Figure 3.2 HPLC chromatograms of EPO-IRS, HSA in dilution buffer (2.5 mg/mL)

and EPO-DP.

The capacity factor (k’) of first peak (HSA) and second peak (EPO) were 3.24 and

5.24, respectively; while the resolution factor was 6.88. The asymmetry of the peak

was found to be 1.29 and 5.29 for EPO and HSA, respectively; while the tailing factor

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parameter was found to be 1.29 and 1.14 for EPO and HSA, respectively. For

replicate injections of EPO-IRS the % RSD of the main peak area was found to be

below 0.7%, and there was no variation in the retention time (less than 0.1 min).

Based on the studied parameters, it was concluded that the EPO and HSA peaks were

well resolved in the developed method and the tailing factor was within limits.

3.3.2.1.2 Method validation

System Suitability:

The chromatographic separation, as explained above was carried out with HPLC to

evaluate the chromatographic parameters. To check the suitability, a known amount

of EPO-IRS was spiked to the dilution buffer and a chromatogram was run.

Representative chromatogram is shown in Figure 3.3, which corresponds to the

chromatographic separation of these substances. The % RSD for the main peak area

of EPO-IRS( measured in triplicate) were found to be below 0.7%, while no variation

in the retention time was observed (less than 0.1 minute). The peaks due to EPO and

HSA were thus considered well resolved.

Figure 3.3: Chromatogram of EPO-DS spiked in dilution buffer containing HSA

2.5mg/mL

min2 4 6 8 10 12 14 16 18

mAU

-100

0

100

200

300

400

500

600

700

13.15

6

One sharp peak of EPO was eluted at 13.1 min along with the HSA peak that was

eluted at 9.0min. The EPO peak was matched with the standard peak of EPO-IRS.

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Specificity

To evaluate possible interfering peaks, two different concentrations of EPO-IRS

(0.04, and 0.1 mg/mL) in dilution buffer and HSA containing dilution buffer were

injected into HPLC. No interference was observed as evidenced by the following

observations:

o No peak was observed in the integration window of the chromatogram for

the sample of mobile phase (blank), HSA in dilution buffer, Dilution

buffer.

o Variation in Retention time of main peak between EPO-IRS and EPO-DP

was less than 0.5 min.

o Variation in terms of % recovery of EPO-IRS spiked in HSA dilution

buffer was less than 5.0 % as compared to EPO-IRS spiked in mobile

phase A.

o There was 0.1 min variation in retention time of main peak of EPO-IRS in

mobile phase as compared to the retention time of the main peak of EPO-

IRS solution (0.1mg/mL).

Linearity and Range

EPO-IRS was used for preparation of different concentrations of EPO-IRS ranging

from 0.028 to 0.130 mg/mL, each containing 2.5 mg/mL HSA. Linearity curves were

plotted for 0.04 and 0.1 mg/mL of EPO-IRS (Figure 3.4).

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Figure 3.4 Linearity curves (HPLC) for (A) 0.04 mg/mL EPO-IRS and (B) and 0.1

mg/mL EPO-IRS

The correlation coefficient, slope, Y-intercept, regression equation of the calibration

curve were determined and are shown in Table A. The percent RSD was found to be

less than 2.0% while the percent recovery was found to be in the range of 97% to

103%.

Accuracy

During accuracy study, we need to consider +20% of the target concentration,

therefore

accuracy was studied using two different sets of three different solutions, containing

0.032, 0.040 and 0.048 mg/mL and 0.08, 0.10 and 0.12 mg/mL of EPO-IRS. Each

solution in its dilution buffer and in the mobile phase was spiked with HSA at a

concentration of 2.5 mg/mL. From Table 3.2 it is evident that the percent recovery

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was found to be more than 95% for 0.04 mg/mL of EPO while the percent recovery

was found to be more than 99% for 0.1 mg/mL of EPO in presence of 2.5 mg/mL of

HSA. The percent RSD was found to be less than 2.0%.

Table 3.2 % recovery of EPO-IRS spiked and control samples of 0.04 mg/mL

Type

Conc. of

EPO-IRS

in mg/mL

Injected

amount of

EPO-IRS (µg)

Avg. value of 3 inj.

Main peak area of

EPO-IRS (mAu*S)

%RSD %Recovery

Spiked

(0.040

mg/mL)

0.032 3.2 975 2.08 96.6

0.040 4.0 1242 0.35 95.7

0.048 4.8 1523 1.38 95.8

Spiked

(0.10

mg/mL)

0.08 8.0 2896 0.76 103.8

0.10 1.0 3642 0.86 102.1

0.12 1.2 4441 0.28 102.1

Control

(0.040

mg/mL)

0.032 3.2 1041 0.92 102.3

0.040 4.0 1339 1.34 102.3

0.048 4.8 1608 0.94 100.6

Control

(0.10

mg/mL)

0.08 8.0 2753 0.24 99.3

0.10 1.0 3566 0.31 100.2

0.12 1.2 4326 0.15 99.7

Precision

Precision was evaluated for intra-day (Repeatability) and inter-day (Intermediate

precision) variation, and different makes of columns.

The repeatability was assessed with six independent sample preparations for each of

the two different system suitability samples (0.04 mg/mL and 0.1 mg/mL of EPO-

IRS) and single injection was injected from each preparation. The percent RSD of

peak area, for each EPO IRS was found to be less than 0.9%.

Inter-day precision was determined by analysis of 0.04 mg/mL of EPO spiked with

2.5 mg/mL of HSA. The precision of the method was evaluated by performing five

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different conditions (n = 30) (Table 3.4) and calculating the relative standard

deviations (RSD). Three replicate injections of system suitability standards prepared

independently were considered for the study. The percent RSD for the main peak

area of EPO-IRS within each set and between different sets was found to be less than

2.0%. The percent recovery of each EPO -IRS was found to be between 95.0% -

105.0% and the maximum variation between sets was found to be 5.0%.

Table 3.3 % RSD of main peak area for the EPO-IRS samples

Sample details Main peak area ( mAu*s) / % recovery of EPO-IRS

%RSD Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6 Avg. value

EPO-IRS

(0.04 mg/mL)

1372 1345 1368 1362 1362 1336 1358 1.03

104.6 102.7 104.3 103.9 103.9 102.1 103.6 0.9

EPO-IRS

(0.1 mg/mL)

3683 3634 3635 3646 3595 3578 3629 1.03

103.2 101.9 101.9 102.2 100.9 100.5 101.8 0.9

Table 3.4 Experimental matrix of intermediate precision

Set 1 Set 2 Set 3 Set 4 Set 5

Equip. ID System-1 System-1 System-1 System-1 System-2

Column Column-1 Column-1 Column-1 Column-2 Column-1

Day 1 2 2 3 3

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84

Table 3.5 % RSD of main peak area for different sets

Sample

details Set No.

Main peak area (mAu*s) %RSD

Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6 Avg. value

EPO-IRS

(0.04

mg/mL)

set-1 1372 1369 1378 1345 1368 1362 1366 0.83

set-2 1323 1314 1299 1308 1299 1281 1304 1.12

set-3 1342 1347 1346 1336 1328 1299 1333 1.36

set-4 1359 1360 1381 1360 1354 1362 1363 0.69

set-5 1363 1358 1357 1338 1351 1354 1353 0.65

EPO-IRS

(0.1

mg/mL)

set-1 3683 3636 3659 3634 3635 3646 3649 0.53

set-2 3578 3577 3535 3528 3499 3501 3536 0.99

set-3 3572 3556 3540 3544 3528 3530 3545 0.47

set-4 3632 3606 3639 3630 3625 3612 3624 0.35

set-5 3676 3671 3671 3670 3658 3648 3666 0.29

Table 3.6 % recovery of EPO-IRS for different sets

Sample

details

Set

no.

% Recovery of EPO-IRS %

RSD Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6 Avg.

value

EP

O-I

RS

(0.0

4m

g/m

L)

set-1 104.57 104.33 104.95 102.71 104.30 103.85 104.12 0.75

set-2 101.22 100.60 99.58 100.19 99.51 98.33 99.90 1.01

set-3 102.51 102.86 102.79 102.06 101.50 99.55 101.88 1.22

set-4 103.65 103.75 103.76 103.76 103.34 103.87 103.69 0.18

set-5 103.96 103.58 103.52 102.20 103.15 103.30 103.28 0.58

EP

O-I

RS

(0.1

mg/m

L)

set-1 103.15 101.94 102.54 101.91 101.93 102.20 102.28 0.48

set-2 100.47 100.46 99.38 99.19 98.45 98.50 99.41 0.90

set-3 100.33 99.91 99.51 99.61 99.19 99.24 99.63 0.43

set-4 101.86 101.19 102.03 101.79 101.67 101.35 101.65 0.32

set-5 102.98 102.85 102.85 102.81 102.52 102.26 102.71 0.26

The % RSD for the main peak area between replicate injections for each EPO-IRS

was less than 2.0 %. Maximum variation between the retention time of main peak for

replicate injections of EPO-IRS was less than 0.2 min. The % recovery for each

EPO-IRS was calculated using respective calibration curve and was found to be

between 95.0% - 105.0%.

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The above results and observations proved that the developed method is precise for

the above mentioned EPO samples when analyzed with respect to, different days,

different instruments and different brands columns (Table 3.5 and 3.6) and hence the

parameter of precision stands validated.

Robustness

Robustness was tested by varying age effect of mobile phase and test samples, column

temperature and mobile phase composition.

Age effect of mobile phase and test samples held for seven days

Freshly prepared samples for system suitability (0.04 mg/mL and 0.1 mg/mL of EPO-

IRS) and those prepared seven days ago were analyzed using both freshly prepared

and seven day old mobile phase. There was not much variation in the results as seen

from Table 3.7, with percent variation from initial day to 7 days being about 5% and

percent RSD being less than 0.4%. There was no difference in retention time and

percent recovery was found to be between 90% and 110%. It is thus recommended to

use freshly prepared sample as well as mobile phase for analysis.

Column temperature effect

Experiments were conducted using system suitability samples with column

temperature variation of + 2ºC from the set temperature (60ºC) and the results are

shown in Table 3.7. The percent RSD was found to be less than 0.7%, with no

variation at lower temperature. However, 5% variation was observed at higher

temperature and + 0.1 minute difference in retention time. The percent recovery was

found to be within acceptable limits (95 – 105%).

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Mobile phase composition

Experiments were conducted using system suitability samples with mobile phase

composition variation of + 20% from the set percentage of TFA (0.1%). The percent

variation between unaltered / initial condition and altered condition for EPO sample

(done in triplicate) as seen from Table 3.7 was found to vary less than 2.0% and there

was no variation in retention time.

Table 3.7 Comparison result for % recovery of EPO-IRS between unaltered / initial

condition and altered condition for 0.1 mg/mL of EPO-IRS

Inj. No.

% Recovery of EPO-IRS peak

Initial

sample

Stored

sample &

mobile

phase

Stored

sample &

fresh

mobile

phase

58ºC

Temp

62ºC

Temp

0.08%

TFA

0.12%

TFA

Inj.1 100.33 89.14 88.74 100.10 99.05 100.33 99.30

Inj.2 99.91 89.45 87.52 100.00 98.94 101.72 98.89

Inj.-3 99.51 89.27 87.80 99.10 98.27 101.17 98.76

Av. value 99.92 89.29 88.02 99.73 98.75 101.07 98.98

Variation

from initial

day/unaltered

condition

(%)

----- 10.63 11.90 0.19 1.17 1.15 0.94

Interpretation:

% RSD for obtained main peak area between replicate injections for each EPO-IRS

with altered and unaltered condition was less than 2.0. Maximum variation between

the retention time of main peak for replicate injections of EPO-IRS for altered and

unaltered condition was less than 0.2 min. Except for stability of sample and mobile

phase, % recovery of each EPO-IRS for altered and unaltered condition was between

95.0 % - 105.0 %, and hence stability of sample and mobile phase can be considered

Chapter 3


87

critical for the method under study. So for routine analysis freshly prepared sample as

well as mobile phase is recommended.

3.3.2.2 UPLC method

3.3.2.2.1 Method development

The basic chromatographic conditions like stationary phase, solvents and UV

detector, employed in HPLC were taken into account while developing the new

UPLC method. The stationary phase C18 was chosen in order to have similar polarity

as that used in HPLC method. The injection volume was scaled down by about 10

fold to that used in HPLC. To get the optimum results, mobile phase flow rate was

kept constant at 0.35 mL/min and column temperature was maintained at 60oC.

The chromatographic separation was achieved as described in Section 3.4.1.

The applied chromatographic conditions permitted a good separation of HSA and

EPO. Different concentrations of EPO-IRS in the range 2.5 to 150 µg with 2.5

mg/mL of HSA were studied and no interference of HSA and other excipients was

observed during the analysis. Representative chromatograms are shown in Figure 3.5.

Chapter 3


88

Figure 3.5 UPLC chromatogram of internal EPO-IRS, HSA in dilution buffer (2.5

mg/mL) & EPO-DP.

AU

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

Minutes

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

The capacity factor (k’) was 2.45 and 3.9 for the first and second peak respectively,

while the resolution factor was 5.35. The asymmetry of the peak was found to be 5.63

and 1.57 for HSA and EPO respectively. Tailing factor was found to be 3.68 and 1.33

for HSA and EPO, respectively. The percent RSD of the main peak area for replicate

injections of EPO-IRS was found to be below 2.0% while no variation in the retention

time was observed (less than 0.1 minutes).

3.3.2.2.2 Method Validation

System Suitability:

The chromatographic separation, as explained above was carried out with UPLC to

evaluate the chromatographic parameters. A sample containing 0.1 mg/mL EPO-IRS

in 2.5 mg/mL HSA in dilution buffer was run and the representative chromatogram is

shown in Figure 3.6, which corresponds to the chromatographic separation of these

EPO IRS

HSA in formulation buffer

EPO Drug Product

Chapter 3


89

substances. The HSA peak was observed at 1.299 min and principal peak of EPO was

obtained at 1.853 min, resolution between both being 5.35. Both peaks were well

resolved in the developed method thus suggesting system suitability. The % RSD for

the main peak area for replicate injections of EPO-IRS was found to be below 2.0%

while no significant variation in the retention time was observed (less than 0.1

minutes).

It was concluded that the developed method is the optimum according to the studied

parameters. The tailing factor to be controlled was within the limits established by

these guidelines. Peak symmetry for the different mobile phase compositions and

different flow rates was compared using the peak asymmetry factor As, measured at

10% of full peak height and the peak-tailing factor (PTF) measured at 5% of full peak

height. Excellent columns give an As value of between 0.95 and 1.1 although values

of < 1.5 are acceptable. PTF values of between 1.0 and 1.4 are also acceptable [29]

Figure 3.6 UPLC chromatogram of EPO-IRS, HSA and Dilution buffer

Chapter 3


90

Specificity

Separation selectivity is the ability of the method to elicit a response specific for the

analyte in the presence of other components/substances that are present or are likely

to be present with the analyte.

To address separation selectivity, 0.1 mg/mL of EPO-IRS in mobile phase (as positive

control), HSA 2.5 mg/mL in dilution buffer, HSA 2.5 mg/mL in mobile phase, 0.1

mg/mL of EPO-IRS with 2.5mg/mL HSA in dilution buffer, mobile phase (Blank),

Milli Q water and dilution buffer were injected into UPLC column.

HSA in dilution buffer and mobile phase was considered as the matrix components.

Interference by the matrix components was evaluated by spiking known amount of

EPO IRS in dilution buffer with HSA. No interference of matrix components was

observed.

Linearity and Range

EPO IRS was used for preparation of different working concentrations ranging from

0.0025 to 0.150 mg/mL, each containing 2.5 mg/mL of HSA. The peak area was

plotted as shown in Figure 3.7.

Chapter 3


91

Figure 3.7 Linearity curve (UPLC) for EPO-IRS

Calibration curves with concentration versus peak area were plotted with blank

subtraction. The correlation coefficient, slopes and Y-intercepts and regression

equation were determined and are shown in Table A. The correlation coefficient was

found to be 0.999. The percent RSD was found to be less than 2.0% while the percent

recovery was found to be in the range of 97% to 103%.

Chapter 3


92

Table 3.8 Result of Linearity experiments:

Conc. of

EPO-IRS in mg/mL

Avg. value of 3 inj.

Main peak area of EPO-IRS

(µV*Sec )

%RSD %Recovery

0.0025 14542.67 3.94 57.60

0.0050 48853.33 0.16 93.52

0.0075 78670.33 0.81 99.85

0.0100 102880.67 1.19 97.72

0.0200 203637.00 0.26 96.38

0.0300 320597.00 0.06 101.02

0.0400 427237.67 0.19 100.91

0.0500 529824.33 0.09 100.08

0.0600 654388.33 0.25 102.98

0.0700 744953.00 0.14 100.47

0.0800 853514.33 0.50 100.71

0.1000 1074215.67 0.39 101.39

0.1200 1255101.33 0.26 98.71

0.1500 1580231.67 0.07 99.41

Accuracy

Accuracy (% recovery) was studied with different working concentrations ranging

from 0.0025 to 0.150 mg/mL of EPO-IRS. Each solution in its dilution buffer was

spiked with HSA at a concentration of 2.5 mg/mL. The percent RSD was found to be

less than 2.0%.

The percentage recovery in the concentration range 0.0075 to 0.15 mg/mL was greater

than 95.0% while the recovery was found to be 93.52% and 57.60% for low EPO

concentrations of 0.0050 and 0.0025 mg/L .

% Recovery was calculated by using the regression equation obtained from linearity

curve of EPO.( y = mx+ c ) where m =slope, c= intercept and y = average area of

particular concentration of EPO-IRS

Chapter 3


93

Back calculated concentration (x)

% Recoveryry

Precision was evaluated by intra-day (Repeatability) and inter-day (Intermediate

precision) variation. Repeatability (five replicates) was assessed independently for

each of the three different concentrations (0.02 mg/mL, 0.04 mg/mL and 0.1 mg/mL).

The percent RSD between areas of all five replicates as shown in Table 3.9 was less

than 1.5% for all dilutions and percent recovery of all five replicates was more than

95%.

Table 3.9 % recovery of five replicates of EPO-IRS.

Table 3.10 Back calculated concentration for five replicates of EPO-IRS:

Sample

mg/mL

Back calculated Conc. of EPO-IRS (diluted)

sample solution (mg/mL)

Average back

calc. conc. Of

EPO

(mg/mL)

%

RSD Inj. 1 Inj. 2 Inj. 3 Inj. 4 Inj. 5

0.02 0.0193 0.0193 0.0194 0.0194 0.0188 0.0192 1.32

0.04 0.0405 0.0405 0.0406 0.0405 0.0403 0.0405 0.28

0.1 0.1018 0.1023 0.1015 0.1014 0.0989 0.1012 1.32

Sample

mg/mL

Area of Principal Peak of

EPO-IRS (diluted) sample solution (µV*Sec) Avg

Area

µV*Sec

%

RSD

%

recovery Inj. 1 Inj. 2 Inj. 3 Inj. 4 Inj. 5

0.02 203085 203689 204137 204133 197869 202583 1.32 96.11

0.04 426584 426986 428143 427039 424861 426723 0.28 101.19

0.1 107354

2

107873

8

107036

7

106922

6 1042460

106686

7 1.32 101.18

Chapter 3


94

The % Recovery of all five replicates was more than 95% for all concentrations of

EPO-IRS studied. The % RSD between areas of the five replicate injection for each

concentration was not more than 2% while the % RSD for retention time of the five

replicate injections of each concentration was not more than 2 %.

From the above results and observations it is established that the developed method

has potential for the quantification of active substance in EPO-DP

Robustness

To determine the robustness of the method, experimental condition (TFA

concentration) was purposefully altered and the resolution between EPO-IRS and

HSA was examined. The TFA concentration was changed between 0.08% and 0.12%

from the standard composition of 0.1% which was originally used and the results are

tabulated in Table 3.11

The percent recovery was found to be between 95% and 105%. The percent RSD for

the area values obtained with altered and unaltered conditions of the parameter was

found to be less than 2.5%, thus indicating that the developed method is robust and

TFA concentration is not a critical parameter.

Table 3.11 Result of % RSD between the altered and unaltered condition for area,

retention time and concentration.- Percentage TFA-0.08% and 0.12%, EPO-IRS-

0.1mg/mL, Injection volume-5μL, Number of injections-2

Chapter 3


95

Sample Unaltered

condition

Altered

condition Average %RSD

TFA concentration : 0.12 % v/v

EPO-

IRS

0.1

mg/mL

Area 1074216 1104096 1089156 1.94

RT 1.869 1.938 1.904 2.56

Conc 0.1021 0.1047 0.1034 1.78

EPO-

IRS

0.1

mg/mL

Area 1074216 1076856 1075536 0.17

RT 1.869 1.841 1.855 1.07

Conc 0.1021 0.1021 0.1021 0.00

3.3.3 COMPARATIVE STUDY OF HPLC AND UPLC PERFORMANCE

The performance parameters of both systems are shown in Table A. The runtime of

UPLC was reduced by 4-fold to that of HPLC. The retention behaviors of HSA and

EPO were similar in HPLC and UPLC columns. As expected, the UPLC method

showed higher efficiency of analysis than HPLC method.

Both RP-HPLC and RP-UPLC methods were demonstrated to be validated for

quantifying EPO in presence of another protein (HSA), which is often present in

medicinal formulations using HSA as stabilizer. The HPLC and UPLC methods were

validated showing satisfactory data for all the parameters tested. The reported UPLC

method was found to be capable of giving faster analysis with good resolution,

accuracy and precision than that achieved with conventional HPLC method. Both the

chromatographic methods were found to be reliable. Since these methods are rapid

and simple, they may be successfully applied to quality control analysis of EPO

formulation containing HSA.

Chapter 3


96

Table A: Results of regression equation/correlation coefficient and both methods

comparative data

Statistical parameter HPLC for 0.04

mg/mL

HPLC for 0.1

mg/mL

UPLC

Linearity & Range

(Conc. in mg/mL)

0.028 to 0.052 0.07 to 0.13 0.0025 to 0.15

Regression Equation y = 36413.9x –

150.967

y = 39168.8x –

357.502

y = 10602431.0x –

725.9877

Correlation coefficient 0.992 0.997 0.999

Total analysis time

(min)

20 20 4

Retention time (min)

For HSA

For EPO

9

13

1.3

1.9

Sample size 100 µL 5 µL

Specificity No interference No interference

Accuracy Recovery more than 95% Recovery more than

97%

Precision RSD less than 2% RSD less than 1.5%

Robustness Method is robust for all tested

parameters except age effect of mobile

phase and test samples

Method is robust for

all tested parameters.

Chapter 3


97

3.4 QUANTIFICATION OF PTH AND METHOD VALIDATION

Human parathyroid hormone (1–84) (hPTH) is a naturally occurring polypeptide

composed of 84 amino acids [30], with overall basic properties (iso-electric point, pI

>9). It has important biological activity as the major regulator of calcium ion

homeostasis [31]. Efficient production methods have been reported through solution

[32] and solid-phase [33] peptide synthesis as well as through recombinant DNA

techniques [34].

Figure 8 Structure of PTH:

Teriparatide (recombinant DNA origin) injection [recombinant human PTH (1–34)

termed as PTH ] is a bone-forming agent for the treatment of osteoporosis. In the

Fracture Prevention Trial (FPT), daily self-injections of teriparatide (20 and 40 µg)

reduced the risk of new vertebral and non-vertebral fractures by 65% and 53%,

Chapter 3


98

respectively, in postmenopausal women with advanced osteoporosis [35]. Once-daily

injection of PTH induced pronounced increase in biochemical markers of bone

turnover [36-41].

Immunoassay is a common technique for measurement of PTH in plasma. The

measurement of PTH and its metabolites has been problematic due to the diversity of

the circulating PTH metabolites, differences in the pharmacokinetic profiles of PTH

and its metabolites and significant differences in specificity and sensitivity of PTH

radioimmunoassay [42-44].

WHO International collaborative study of the proposed 1st international standard for

recombinant human PTH (1-84) was done by RP-HPLC method in different

laboratories [45]. Liquid chromatographic studies on separation of ten PTH amino

acids were carried out using normal phase untreated silica gel plate, C-18 RP

precoated plates and RP-HPLC by R. Bhushan et al. [46]. Separation,

characterization and biological activity of PTH oxidized at methionine8 and

methionine 18* was studied by A.L. Frelinger et al. [47]. PTH was oxidized with

hydrogen peroxide and the biological activity of oxidation products was studied by

Nabuchi et al. [48]. Methionine oxidation in PTH was also studied by Y. Nabuchi et

al. by using RP-HPLC [49].

The PTH (1-34) formulation contains meta-cresol as antimicrobial preservative,

which may interfere with OD280 UV detector of HPLC for either PTH or m-cresol as

well as with colorimetric assays . An RP-HPLC/UPLC method which can specifically

measure the protein component of Active Pharmaceutical Ingredient (API) with

separation of meta-cresol from protein peaks; will be suitable for quantitation of the

active substance in the presence of meta-cresol. The objective was to develop

Chapter 3


99

methods, using RP-HPLC and UPLC techniques to enable quantification of PTH in

medicinal formulations containing meta-cresol as well as a method to quantify meta-

cresol.

PTH formulation (drug product ) contains 250 µg/mL of PTH(API), 3mg/mL meta-

cresol; 45.4mg/mL mannitol; 0.1mg/mL sodium acetate and 0.41mg/mL glacial acetic

acid in water for injection

Abbreviations used:

PTH – Parathyroid Hormone

PTH IRS – PTH (1-34) Internal Reference Standard

DS (API) – Drug Substance (Active Pharmaceutical Ingredient)

PTH-DS – PTH Drug Substance

DP – Drug Product

PTH-DP – PTH Drug Product

RMP – Reference Medicinal Product (Innovator Product – Forteo). It is drug product

rHu – Recombinant Human

EP – European Pharmacopoeia

3.4.1 EXPERIMENTAL

Materials, reagents and chemicals

HPLC grade acetonitrile and methanol were purchased from Merck, tri-fluoro-acetic

acid (TFA) was purchased from Sigma Aldrich. Ultra pure water was obtained using

Milli-Q® UF-Plus (Millipore) system; meta-cresol was obtained from J.T. Baxter/

Hedinger. Reference Medicinal Product (herewith termed as RMP) having a

concentration of 250 µg/mL and PTH-IRS obtained from Intas Biopharmaceuticals

were used for preparation of standards in all experiments. All other chemicals such

Chapter 3


100

as mannitol, sodium acetate and glacial acetic acid were of the highest purity

available.

Preparation of mobile phase, dilution buffer and standard

Mobile phase ‘A’ consisted of 0.1% (v/v) TFA in Milli Q water and mobile phase ‘B’

consisted of 0.1% (v/v) TFA in acetonitrile. Dilution buffer containing 3 mg/mL

meta-cresol; 45.4 mg/mL mannitol; 0.1 mg/mL sodium acetate and 0.41 mg/mL

glacial acetic acid in “Milli Q water” was prepared and used so as to have a matrix

similar to PTH formulation. Diluted PTH-IRS was prepared by using 400 µg/mL of

PTH IRS using mobile phase A. Oxidized form of PTH was prepared by adding 4.0

µL of diluted 0.25 % H2O2 to 62.6 µL of PTH IRS (0.4 mg/mL) and mixing well. The

solution was incubated for 40 minutes at room temperature and then quenched with

37.4 µL of 50 mg/mL methionine [50]. All dilutions were made using calibrated

digital micro-pipettes.

Chromatographic condition

Agilent LC system (1100 and 1200 series) equipped with an injection valve

(quaternary), 210 UV detector and Chemstation software was used for HPLC method.

A reversed-phase C18 column (2.1mm ID × 100mm L, porosity 300ºA, particle size

3µm) with guard column (reversed-phase C18 column of 2.1mm ID × 12.5mm L,

porosity 300ºA, particle size 5µm) was used for separation. To get the optimum

results, mobile phase with a flow rate of 0.3mL/min was used and column

temperature was maintained at 60oC. The gradient programme for mobile phase was

optimized using a timed gradient programme T(min)/mobile phase A (%): 0/80, 6/80,

26.1/45, 28/0, 31/0, 31.5/80, and 40/80.

Chapter 3


101

Waters LC system (ACQUITY) equipped with an injection valve (binary), 215UV

detector and Empower software was used for RP-UPLC method. Reversed-phase C8

column (2.1mm ID × 12.5mm L, porosity 300ºA, particle size 5µm) was used for

separation. To get the optimum results, mobile phase flow rate was kept constant at

0.4mL/min, column temperature at 60oC. The gradient programme for mobile phase

was optimized using a timed gradient programme T(min)/mobile phase A (%): 0/80,

1.2/80, 4.8/0, 5/80, and 6/80.

3.4.2 RESULTS AND DISCUSSION

3.4.2.1. RP-HPLC Method

3.4.2.1.1. Method development

Initially, the gradient HPLC conditions were optimized for determination of PTH IRS

in presence of meta-cresol. The chromatographic separation was achieved by

applying chromatographic conditions described in Section 3.5.1

The applied chromatographic conditions permitted a good separation of meta-cresol

and PTH at different concentrations of PTH. No interference of other excipients or

oxidized impurities was observed as shown in Figure 3.9 and Figure 3.10.

Chapter 3


102

Figure 3.9 Overlapped HPLC chromatograms of (A) mobile phase (as blank), (B)

dilution buffer, (C) PTH-DP

Mobile phase, dilution buffer, PTH-DP injected into HPLC separately

A) Mobile phase – 0.1% trifluoroacetic acid (TFA) in MilliQ water and 0.1%

TFA in acetonitrile

B) Dilution buffer (without meta-cresol) – 45.4 mg/mL mannitol; 0.1 mg/mL

sodium acetate and 0.41 mg/mL glacial acetic acid in Milli Q water

C) PTH–DP – 0.250 mg/mL of PTH-DS and 3 mg/mL meta-cresol

Chapter 3


103

Figure 3.10 HPLC chromatogram of oxidized form of PTH

min12 14 16 18 20 22 24 26

mAU

-50

0

50

100

150

200

250

300

VWD1 A, Wavelength=210 nm (D:\HPLC06\DATA\DATA2009\AUG09\P8AG0931\7\P8000015.D)

14.

122

15.

054

15.

512

15.

771

16.

062

16.

398

17.

199

4.0 µL of diluted 0.25% H2O2, added to PTH-DS (0.4 mg/mL), mixed well, incubated

for 40 minutes at room temperature, and then quenched with 37.4µL of 50 mg/mL

methionine.

The capacity factor (k’) of the first peak (meta-cresol) and second peak (PTH) were

1.60 and 11.5, respectively; while the resolution factor was 6.88. The asymmetry of

the peaks for meta-cresol and PTH were found to be 0.26 and 1.06, respectively;

while the tailing factor parameters for meta-cresol and PTH were found to be 3.62 and

1.36, respectively. Based on the studied parameters, it was concluded that the

developed method is optimum. PTH, oxidized impurities and meta-cresol peaks were

well resolved and the tailing factor was within limits.

Chapter 3


104

3.4.2.1.2. Method validation

System suitability

To verify the interference and resolution, PTH-DP, diluted PTH-DP and oxidized

PTH-DS were injected into HPLC as mentioned below and the observations are

tabulated in Tables 3.13 and 3.14:

Reference solution-1: Innovator product (commercial available in market) Forteo

(RMP)(labeled concentration is 0.25 mg/mL) has been used as reference solution 1.

Reference solution-2: Reference solution -1 was diluted to get 0.005 mg/mL of PTH

in mobile phase A.

Reference solution-3: PTH-IRS (0.4 mg/mL) was used as the stock solution for

preparation of reference solution 3 (Oxidized solution). For preparing 0.25% H2O2,

the commercially available 50% H2O2 was diluted 200 times. 5 µL of H2O2 was added

to 995µL of MilliQ water and mixed well. 4µL of the diluted H2O2 solution was

added to 62.6 µL of PTH-IRS (0.4 mg/mL) and mixed well. The solution was

incubated for 40 minutes at room temperature and then quenched with 37.4µL of 50

mg/mL methionine. Thus the PTH-IRS gets diluted ~1.6 times and the final

concentration of the sample is 0.25 mg/mL

Maximum variation of retention time between principal peak of reference solution 1

and 2 was found to be 0.1 minute (retention time is about 16.3 min.). The %

recovery of the reference solution 2 when compared to 2% of the total area obtained

with reference solution 1 was found to be 92.3%. The % RSD of the 2% solutions

(reference solution 2) ( for three replicate measurements ) is 1.9%. The resolution

between the principal peak and oxidized peak nearest to the principal peak is 3.9. The

variation in retention time of principal peak of the standard reference solutions 1 and

Chapter 3


105

2 is 0.1 minute. The % RSD for the total areas of the standard (Reference solution

1),for three measurements is 0.2%. The %RSD for the total areas of the of the

standard (Reference solution 2) for two measurements is 1.9%. (Refer table 3.14)

Specificity

To evaluate possible interfering peaks, PTH-IRS (250µg/mL) in mobile phase (as

positive control); API; drug product (to verify the separation of interested protein

from other components) and oxidized PTH-DS; (to confirm the separation of oxidized

forms of protein from the interested protein) were injected into HPLC and no

interference was observed as shown in Figure3.10.

Retention time of Forteo (RMP) was found to match exactly with the PTH-IRS and

PTH-DP. Four very well resolved oxidized impurities were observed in the range of

11 to 20 minutes in the chromatogram of PTH-DS. There was no peak observed in the

chromatogram of blank (mobile phase) and dilution buffer without meta cresol. There

were 5 peaks observed in dilution buffer with meta cresol.

In the oxidised spike sample, peak of PTH was eluting after the main peak of Forteo

(RMP), and the resolution obtained was more than 1.0. The %RSD between average

purity percentage of 3µg, 4µg and 5 µg of 0.25 mg/mL PTH-IRS diluted in mobile

phase and dilution buffer without meta cresol was found to be 0.12 and 0.07%

respectively. The %RSD between average purity percentage of 3µg, 4µg and 5 µg of

0.25 mg/mL PTH-DP diluted in mobile phase and dilution buffer with meta-cresol

was found to be 0.17 and 0.08% respectively. The %RSD between total areas of

individual preparation of each amount of 3µg, 4µg and 5 µg of DS & DP diluted in

mobile phase and dilution buffer was found to be not more than 2%. The % RSD

between average purity percentage of 3µg, 4µg & 5 µg of PTH-DP diluted in mobile

Chapter 3


106

phase and dilution buffer with metacresol was found to be 0.02%, 0.03% & 0.24%

respectively.

Linearity and Range

PTH IRS was used for preparation of different concentrations ranging from 100 to

312µg/mL. Linearity curve was plotted for peak area responses versus concentration

of PTH and is shown in Figure3.11.

Figure 3.11 Linearity curve (HPLC) for PTH

PTH-IRS (400 µg/mL) was diluted with mobile phase A for preparation of different

concentrations ranging from 100 to 312 µg/mL injected separately.

The correlation coefficient, slope, Y-intercept, regression equation of the calibration

curve was determined and the results are shown in Table B. The percent RSD was

found to be less than 2.0% while the percent recovery was found to be in the range of

97% to 103%.

Linearity curve (100 µg/mL to 312µg/mL of PTH IRS)

y = 82.168x - 47.221

R 2 = 0.9995

0

5000

10000

15000

20000

25000

30000

0 50 100 150 200 250 300 350 Concentration (µg/mL)

Average area (mAu*s)

Chapter 3


107

Table 3.13 Area / % Recovery and % RSD

Injected

Amount

(µg)

Conc.

mg/mL

Total

Area 1

(Soln1)

Total

Area 2

(Soln2)

Total

Area 3

(Soln3)

Avg.

Total

area

SD %

RSD

Back

calculated

conc.

(mg/mL)

%

recove

ry.

0.04 0.002 84.5 87.5 72.4 81.4 7.9 9.8 0.04 104.6

0.05 0.0025 117 118 120 118.3 1.5 1.3 0.05 102.6

0.08 0.004 157 195 197 196 1.4 0.7 0.07 89.0

0.1 0.005 315 322 318 318 3.5 1.1 0.10 102.6

0.5 0.025 1936 1959 1964 1953 14.9 0.8 0.50 99.6

1 0.05 3799 4118 4065 3994 170.9 4.3 0.99 99.4

2 0.1 7931 7901 8074 7968 92.4 1.2 1.96 98.0

3 0.15 12318 12763 12389 12490 239.0 1.9 3.06 101.9

4 0.2 16374 16428 16638 16480 139.4 0.8 4.03 100.7

5 0.25 20458 20470 20447 20458 11.5 0.1 4.99 99.9

6.25 0.25 25570 25491 25501 25520 43.0 0.2 6.22 99.6

Accuracy

Accuracy was studied by spiking PTH in the range of 200, 250 and 312 µg/mL in the

mobile phase and calculating the percent recovery. The percent recovery as seen from

Table 3.14 was found to be in the range of 95% to 105%. The percent RSD was

found to be less than 2.0%.

Chapter 3


108

Table 3.14 Area / % Recovery and % RSD

Conc. of

PTH

Amount

of

protein

injected

(µg)

Area of main peak (mAu)

Average %

RSD

Back

calculated

amount

(µg)

%

Recovery

from the

graph Inj.1 Inj.2 Inj.3

0.002

(0.8%) 0.04 84 76 76 79 5.75 0.05 116.5

0.0025

(1.0%) 0.05 92 93 91 92 0.66 0.05 99.3

0.004

(1.6%) 0.08 169 160 172 167 3.80 0.07 83.5

0.2

(80%) 4 15377 15384 15404 15388 0.09 4.14 103.4

0.25

(100%) 5 18845 18883 18880 18869 0.11 5.12 102.4

0.25

(125%) 6.25 22727 22783 22825 22778 0.22 6.23 99.7

Precision

Precision was evaluated for intra-day (Repeatability) and inter-day (Intermediate

precision) variation, and for different columns. Intra-day study was determined by

using six independent preparations of the PTH-DS (250 µg/mL) and PTH-DP (250

µg/mL) as shown in Table 3.15. The percent RSD of main peak area was found to be

less than 0.5%. Inter-day precision was determined by performing five different

conditions along with five replicates for each condition which is equivalent to n = 25

(5 × 5) as shown in Table 3.17. The percent RSD of the main peak area was found to

be less than 0.5% within each set and less than 2.0% between different sets. The

percent recovery was found to be between 95.0% - 105.0 % and the maximum

variation between sets was found to be less than 5.0%.

Chapter 3


109

Table 3.15 Total area of PTH-DS and PTH-DP

Sample Details

Total area of PTH-IRS (DS) and PTH-DP

(mAu*s) Averag

e

%

RS

D Soln.1 Sol

n.2 Sol

n.3 Sol

n.4 Sol

n.5 Sol

n.6

0.25 mg/mL

DS 18845 18845 18845 18845 18845 18845

18842 0.2

0.25 mg/mL

DP 18364 18364 18364 18364 18364 18364 18368 0.2

Table 3.16 Percent purity of PTH–DS and PTH-DP.

Sample Details Purity percentage of PTH-DS) and PTH-DP

Average

Percent

purity

%

RS

D Soln.1 Soln.2 Soln.3 Soln.4 Soln.5 Soln.6

0.25 mg/mL

DS 98.35 98.35 98.35 98.35 98.35 98.35 98.0 0.03

0.25 mg/mL

DP 94.16 94.16 94.16 94.16 94.16 94.16 94.0 0.1

Table 3. 17 Experimental matrix of intermediate precision

Set 1 Set 2 Set 3 Set 4 Set 5 Set 6

Equip. ID System-1 System-1 System-1 System-2 System-1 System-2

Column Column-1 Column-1 Column-1 Column-1 Column-1 Column-2

Day Day-1 Day-1 Day-2 Day-2 Day-2 Day-2

Robustness

The robustness is a measure of its capacity to remain unaffected by small, but

deliberate variations in method parameters and provides an indication of its

robustness during normal usage. Robustness was tested using three variables: flow

rate, column temperature, and mobile phase composition.

Chapter 3


110

Flow rate

Experiments were conducted using system suitability samples of concentrations

0.005 and 0.25 mg/mL prepared from Forteo (RMP), with flow rate variation of +10%

from the set flow rate (0.3 mL/min). The percent RSD was found to be less than 2%,

with no variation and +0.1 minute difference in retention time but during lower flow

rate, higher percentage recovery (~ 111%) was obtained as compared to higher flow

rate, we found lower percentage of recovery (~ 93%). Based on recovery, it was

concluded that flow rate is critical parameter.


Experiments were conducted using the same system suitability samples (as used in

flow rate studies) with column temperature variation of +5ºC from the set temperature

(60ºC). The percent RSD was found to be less than 2%, with no variation and +0.1

minute difference in retention time. The percent recovery was found to be within

acceptable limits (95%-105%) and hence column temperature was not considered to

be critical parameter.


Experiments were conducted using system suitability samples( same as used in flow

rate) with mobile phase composition variation of + 20% from the set percentage of

TFA (0.1%). Results for triplicate injections (% variation) between unaltered / initial

condition and altered condition for PTH sample were found to vary less than 2.0%

without variation in retention time and hence mobile phase composition was not

considered to be critical parameter.

Chapter 3


111

3.4.2.2. UPLC method

3.4.2.2.1. Method development

The basic chromatographic conditions like stationary phase, solvents and UV

detector, employed in HPLC were taken into account while developing new UPLC

method. The stationary phase C8 was chosen in order to have similar polarity to that

used in the method developed for HPLC. The injection volume was scaled down by

about 5 fold as used in HPLC. To get the optimum results, mobile phase flow rate was

kept constant at 0.4 mL/min and column temperature was maintained at 60oC. The

chromatographic separation was achieved as described in Section 3.5.1

The applied chromatographic conditions permitted a good separation of meta-cresol

and PTH at different concentrations of PTH. No interference of other excipients or

other oxidized impurities was observed during the analysis and are shown in Figure

3.12-3.14.

Figure 3.12 Overlapped UPLC chromatograms of (A) mobile phase, (B) dilution

buffer and (C) PTH-DS (without meta-cresol)

Mobile phase, dilution buffer and PTH into HPLC separately

A) Mobile phase – containing 0.1% trifluoroacetic acid (TFA) in MilliQ water

Chapter 3


112

and 0.1% TFA in acetonitrile

B) Dilution buffer (without meta-cresol) – containing 45.4 mg/mL mannitol; 0.1

mg/mL sodium acetate and 0.41 mg/mL glacial acetic acid in Milli Q water

C) PTH–DS – containing 0.250 mg/mL of PTH in Milli Q water

Figure 3.13 UPLC chromatograms of (A) meta-cresol and (B) PTH-DP (with meta-

cresol)

PTH–DP – containing 0.250 mg/mL of PTH and 3 mg/mL meta-cresol

Chapter 3


113

Figure 3.14 UPLC chromatogram of oxidized form of PTH

4.0 µL of diluted 0.25% H2O2, added to PTH-IRS (0.4 mg/mL), mixed well,

incubated for 40 minutes at room temperature, and then quenched with 37.4µL of 50

mg/mL methionine

The capacity factor (k’) of the main peak (PTH) was 11.60; while tailing factor was

found to be 1.24. It can be thus concluded that PTH, oxidized forms of PTH and

meta-cresol peaks were well resolved in the developed method and the tailing factor

was within limits.

3.4.2.2.2. Method Validation

System suitability

Two types of system suitability were evaluated by analyzing PTH-IRS for the

respective parameters through-out the validation study.

System suitability A – The RSD between the areas of the first three injections should

not be more than 1 % while RSD between the areas of peak in five injections should

not be more than 1 %.

Chapter 3


114

System suitability B – The RSD between the areas of the first three injections should

not be more than 1 %. The back calculated concentration based on average area of

first three injections as well as last two injections of the standard sample should not

show variation more than 5% of the pre-determined concentration (150µg /mL) as in

the calibration curve.

System suitability A was evaluated with linearity and range and B was evaluated with

other validation parameters.

Specificity

To evaluate possible interfering peaks, diluted PTH-IRS (150µg/mL) in mobile phase

(as positive control); API; PTH-DP (to verify the separation of interested protein from

other components) and oxidized API; oxidized PTH-DP (to confirm the separation of

oxidized forms of protein from the interested protein) were injected into UPLC and no

interference was observed as seen from Figure 3.14. From Table 3.18 it is evident

that the percent variation in peak areas of PTH-DS and oxidized PTH-DS was found

to be less than 2%.

Chapter 3


115

Table 3.18 – Area / % variation

Sample Name

Average total area

of main peak

(mAu*S)

Conc. of PTH

(µg / mL)

%

Variation

PTH-DS 1681222 145.5 1.5

Oxidized PTH-DS 1654234 143.3

PTH-DP 1686672 146.0 1.6

Oxidized PTH-DP 1659430 143.7

Linearity and Range

PTH RS and samples were chromatographed using the set chromatographic

conditions. Linearity curve was plotted using 50 to 300 µg/mL of PTH and is shown

in Figure 3.15.

Figure 3.15: Linearity curve (UPLC) for PTH:

PTH-IRS (400 µg/mL) was diluted with mobile phase ‘A’ for preparation of different

concentrations ranging from 50 to 300 µg/mL, injected separately.

The linearity of peak area responses versus concentration for PTH was studied and

correlation coefficient, slopes and Y-intercepts and regression equation were

Chapter 3


116

determined and the results are shown in Table B. The correlation coefficient was

found to be 0.999. It is also observed from Table 3.19 that the percent RSD was

found to be less than 2.0% while the percent recovery was found to be in the range of

98% to 105%.

Table 3.19 % RSD and % Recovery for the area of principal peak of PTH-IRS.

Conc. of

PTH

(µg/mL)

Area of main peak of PTH

(mAu*s) Average

area

(mAu*s)

%

RSD

Back

calculated

conc.

(µg/mL)

%

recovery Prep:1 Prep:2 Prep:3

50 -- 548622 540801 544712 1.02 52.4 104.8

100 1115814 1135939 1123823 1125192 0.90 100.0 100.0

150 1719512 1699157 1726950 1715206 0.84 148.3 98.9

200 2317483 2321624 2322369 2320492 0.11 197.9 99.0

250 2924020 2947362 2899222 2923535 0.82 247.3 98.9

300 3611137 3671138 3565685 3615987 1.46 304.1 101.4

Accuracy

Accuracy was studied using six different solutions, containing 50, 100, 150, 200, 250

and 300 µg/mL of PTH. Three replicates of each solution were spiked in the mobile

phase. (n= 6 × 3 = 18) and the results are tabulated in Table 3.19. The percent

recovery was found to be in the range of 95% to 105%. The percent RSD was found

to be less than 2.0%. .

Precision

Intra-day (Repeatability) and inter-day (Intermediate) precision were evaluated using

different equipment and different lots of UPLC columns of same makes. Intra-day

precision studies were performed by injecting six independent preparation of the

PTH-IRS (150 µg/mL). The percent RSD of main peak area was found to be less than

0.5 % as seen from Table 3.20. Inter-day precision study was done under five

different conditions along with six replicates for each condition which is equivalent

Chapter 3


117

to n = 30 (5 × 6) as shown in Table 3.21 and the results are tabulated in Table 3.22.

The percent RSD of the main peak area was found to be less than 0.5% within each

set and less than 3.0% between different sets. The percent recovery was found to be

between 95.0% - 105.0% and the maximum variation between sets was found to be

less than 5.0%.

Table 3.20 Peak area (diluted PTH-IRS-0.15 mg/mL) and retention time for the

principal peak of InRS.

Sample

preparation

Area of main peak of PTH-

IRS ( mAu*s)

Set-1 Set-2

Prep-1 1679634 1.889

Prep-2 1671084 1.888

Prep-3 1663891 1.888

Prep-4 1675372 1.891

Prep-5 1667722 1.888

Prep-6 1668911 1.888

Avg. value 1671102 1.889

RSD 0.34 0.06

Table 3.21 Experimental matrix of intermediate precision


Equip. ID System-1 System-1 System-2 System-2 System-1


Day Day-1 Day-2 Day-3 Day-4 Day-4

Chapter 3


118

Table 3.22 Intermediate precision study data

Sample

preparation

Area of main peak of PTH-IRS ( mAu*s)

Set-1 Set-2 Set-3 Set-4 Set-5

Prep-1 1679634 1677355 1392999 1635180 1689759

Prep-2 1671084 1667891 1398166 1643618 1677671

Prep-3 1663891 1676642 1400129 1650774 1688277

Prep-4 1675372 1674680 1395728 1658175 1684267

Prep-5 1667722 1682425 1396970 1641093 1675686

Prep-6 1668911 1677319 1391947 1644555 1692449

Avg. value 1671102 1676052 1395990 1645566 1684685

RSD (%) 0.34 0.28 0.22 0.49 0.40

Robustness

The robustness is a measure of its capacity to remain unaffected by small, but

deliberate variations in method parameters and provides an indication of its

robustness during normal usage. Robustness was tested using three variables, age

effect of mobile phase and test samples, column temperature and mobile phase

composition.

Age effect of mobile phase and test samples held for seven days

Freshly prepared samples for system suitability (150 µg/mL of PTH) and those

prepared seven days ago were analyzed using both freshly prepared and seven days,

old mobile phase. There was not much variation in the results, with percent variation

from initial day to 7 days being about 5% and percent RSD being less than 1.0%.

There was no difference in the retention time and percent recovery was found to

be in between 95% and 105%, indicating that age of the mobile phase was not a

critical parameter.

Chapter 3


119


Experiments were conducted using system suitability samples with column

temperature variation of + 5ºC from the set temperature (60ºC). The percent RSD was

found to be less than 2%, with variation of + 0.1 minute in the retention time. The

percent recovery was found to be within acceptable limits (95%–105%) suggesting

that the variation in the results was within acceptable limits for all the parameters

under study and indicating that column temperature was not a critical parameter.


Experiments were conducted using system suitability samples with mobile phase

composition variation of + 20% from the set percentage of TFA (0.1%). Results for

triplicate injections (% variation) between initial condition and altered condition for

PTH sample were found to vary less than 2.0% and there was no variation observed in

the retention time, suggesting that the mobile phase composition was not a critical

parameter.

Chapter 3


120

Table 3.23 % RSD for main peak area and back calculated concentration of PTH-IRS

for all sets

Injection

details

Main peak area of PTH-IRS (mAu*s)

Initial

sample

Stored

sample

&

mobile

phase

Stored

sample &

fresh

mobile

phase

55ºC

Temp

65ºC

Temp

0.08%

TFA

0.12%

TFA

Inj.1 1667980 1689027 1681233 1736020 1683363 1697866 1670305

Inj.2 1668104 1682446 1679217 1733345 1677123 1689116 1669403

Inj.3 1672415 1680431 1682088 1733179 1670967 1680852 1674829

Inj.4 1661187 1681334 1678649 1732007 1664147 1677127 1682958

Inj.5 1636550 1679619 1678866 1735579 1780984 1675052 1685501

Avg. value for

first 3 inj. 1669500 1683968 1680846 1734181 1677151 1689278 1671512

% RSD for

first 3 inj. 0.15 0.27 0.09 0.09 0.37 0.50 0.17

Back

calculated

Conc. of 5

Injections in

µg/mL

143.9 145.6 145.4 149.9 146.7 145.8 145.2

Variation

from

predetermined

conc.

(150 µg /mL)

4.1 2.9 3.1 0.1 2.2 2.8 3.2

3.4.3 COMPARATIVE STUDY OF HPLC AND UPLC PERFORMANCE

The performance parameters of both systems are shown in Table B. The runtime of

UPLC was reduced by 7-fold to that of HPLC. The retention behaviors of meta-

cresol, PTH, and oxidized impurities were similar in HPLC and UPLC columns. As

expected, the UPLC method showed higher efficiency of analysis than HPLC method.

Both RP-HPLC and RP-UPLC methods were demonstrated to be validated for

Chapter 3


121

quantifying PTH respectively in presence of other excipients and oxidized impurities

of PTH. The HPLC and UPLC methods were validated showing satisfactory data for

all the parameters tested. The UPLC method was found to be capable of giving faster

analysis with good resolution, accuracy and precision than that achieved with

conventional HPLC method. Both the chromatographic methods described here were

found to be reliable for quantifying PTH. Since these methods are rapid and simple,

they may be successfully applied to quality control analyses of finished (formulated)

product in presence of meta-cresol.

Chapter 3


122

Table B Results of regression equation/correlation coefficient and both methods

comparative data

Statistical parameter RP-HPLC UPLC

Linearity and range

Concentration (µg/mL)

100 – 300 50 – 300

Regression Equation y = 82.168x – 47.221 y = 12203.8226x – 94815.0889

Correlation coefficient (R2) 0.999 0.999

Total Analysis time (min) 40 6

Retention time (min)

For meta-cresol

For PTH

3.5

16.4

0.3

1.9

Flow rate (mL/min) 0.3 0.4

Column C18

(2.1 x 100 mm)

3µ, 300oA

C8

(2.1 x 12.5 mm)

5µ, 300oA

Column condition C18, 210 UV

C8, 215 UV

Method Gradient Gradient

Sample size 20 µL 4 µL

Specificity No interference No interference

Accuracy Recovery between

95% - 105%

Recovery between

95% - 105%

Precision RSD <3.0% RSD <1.0%

Robustness Yes Yes

Chapter 3


123

3.5 QUANTIFICATION OF META-CRESOL AND METHOD VALIDATION

Introduction

Meta-cresol is widely used as bactericide in the biotechnological processing of

pharmaceuticals; preservative in pharmaceutical formulations (injection solutions of

insulin, somatropin, parathyroid hormone); pesticide for the treatment of the stems of

fruit trees and plants. Exposure of humans is possible through the use of m-cresol as

a preservative in pharmaceutical injection solutions.

Meta-cresol, para-cresol and m/p-cresol mixtures are absorbed across the respiratory

and gastrointestinal tracts and through the skin, and are distributed throughout the

body. The primary metabolic pathway for all cresol isomers is conjugation with

glucuronic acid and inorganic sulfates. All isomers are mainly eliminated by renal

excretion in form of above-mentioned conjugates. The oral LD50 of undiluted m-

cresol in rats was 242 mg/kg bw. Clinical signs include hypoactivity, salivation,

tremors, and convulsions. Neither mortality nor clinical signs of toxicity were seen

following exposure to saturated vapour concentration of either m-cresol or p-cresol.

Inhalation of aerosols may however cause death, and mean lethal concentrations in

rats were reported to be 29 mg/m³ for p-cresol and 58 mg/m³ for m-cresol [51].

Reaction to meta-cresol in commercial preparations of insulin to humans was reported

by Dennis et al.[52].

The analysis of cresol- like chemicals in use for a long period of time has evolved

from a number of nonspecific colorimetric methods to more selective separation

techniques using gas chromatography (GC) or high performance liquid

chromatography (HPLC) [53-55].

Chapter 3


124

The objective was hence to develop a rapid and simple RP-HPLC method with UV

detection, useful for routine quality control of m-cresol in parathyroid hormone

formulations (PTH).

To obtain the best chromatographic conditions, the mobile phase composition, column

temperature and flow rate were optimised. The flow rate was varied from 0.8 mL

min−1

to 1.2 mL min−1

. The column temperature was varied between 22oC to 30

oC

and the analysis at 30◦C was preferred on the basis of improved peak symmetry and

resolution. The % of mobile phase was varied + 2% from the set parameters i.e.

60%+2% (58% to 62%). Isocratic chromatographic conditions were optimized for

determination of meta-cresol in PTH pharmaceutical product. The applied

chromatographic conditions permitted a good separation of meta-cresol and PTH at

different concentrations of meta-cresol. No interference of other excipients was

observed as shown in Figures 3.16-3.17.

Figure 3.16: Sample Chromatogram of principal peak of dilution buffer, meta-cresol

standard and mobile phase:

Meta-cresol ref. std.

Mobile phase

Dilution buffer

Chapter 3


125

Figure 3.17: Sample Chromatogram (overlap) of principal peak of PTH API & meta-

cresol standard:

3.5.1 EXPERIMENTAL

Material, Reagent & Chemicals:

HPLC grade acetonitrile and methanol were purchased from Merck; tri-fluoro-acetic

acid was purchased from Sigma Aldrich. Ultra pure water was obtained using Milli-

Q® UF-Plus (Millipore) system; meta-cresol was obtained from J.T. Baxter/ Hedinger

and was used for preparation of different dilutions; PTH-DS having concentration of

400 µg/mL was used for diluted sample of PTH; PTH-DP was used as test sample.

All chemicals i.e. mannitol, sodium acetate and glacial acetic were of the highest

purity available.

Preparation of standard, mobile phase and dilution buffer:

Dilution buffer: Buffer containing 3mg/mL meta-cresol; 45.4mg/mL mannitol;

0.1mg/mL sodium acetate and 0.41mg/mL glacial acetic acid in Milli Q water was

prepared. It is similar as excipients used for PTH formulation.

Diluted PTH standard: PTH (400 µg/mL) was used for preparation of different

diluted samples.

rHu PTH DS

Meta-cresol ref. std.

Chapter 3


126

Meta-cresol standard: 3 mg/mL was used for preparation of different dilutions.

Mobile phase consisted of 0.1% v/v TFA in 60% methanol.

All dilutions were made using calibrated digital micro-pipettes.

Chromatographic condition:

LC system equipped with an injection valve (quaternary), 217 UV detector and

Chemstation software was used. A reversed-phase Jupiter C18 column (4.6mm ID ×

250mm L, porosity 300ºA, particle size 5µm) with guard column (reversed-phase C18

column of 4.6mm ID × 12.5mm L, porosity 300ºA, particle size 5µm) was used for

separation. To get the optimum results, mobile phase with a flow rate of 1.0mL/min

was used and column temperature was maintained at 30oC. The isocratic programme

for mobile phase was optimized for 12 minutes.

3.5.2 RESULTS AND DISCUSSION

3.5.2.1 Method development:

The capacity factor (k’) of the first peak (meta-cresol) and second peak (PTH) were

3.24 and 5.24, respectively; while the resolution factor was 6.88. The asymmetry of

the peak for meta-cresol and PTH were found to be 1.29 and 5.29, respectively; while

the tailing factor parameter for meta-cresol and PTH was found to be 1.29 and 1.14,

respectively. For replicate injections of meta-cresol standard; the % RSD of the main

peak area was found to be below 0.7%, and there was insignificant variation in the

retention time (less than 0.1 min).

The PTH, and meta-cresol peaks were thus found to be well resolved and the tailing

factor was within limits. As available PTH formulations in the market contain

100µg/mL of meta cresol the concentration range of m-cresol was selected from

Chapter 3


127

75µg/mL to 120 µg/mL. Detection limit (LOD) and quantification limit (LOQ) were

not done for the study.

Usage of different column brands: –

Regarding the chromatographic procedure, different brands of reverse phase C18

columns were used (Jupiter column and Grace Vydac column) and compared in terms

of percentage variation of principal peak area of meta-cresol standard. Experiments

were conducted using system suitability samples. Percentage variation between

principal peak of meta-cresol was not more than 5% in all samples when compare to

the area of principal peak of meta-cresol from specificity samples. Retention time of

principal peak of meta-cresol was found to be around 5.3 minutes and 3.9 minutes

whereas principal peak of PTH was found to be around 10.9 minutes and 4.7 minutes

on Jupiter and Grace Vydac column respectively. Principal peak in both samples was

separated by base to base while overlapping their chromatograms. Integrable peak

was found at the retention time of the principal peak of meta-cresol in the API

dilution buffer without meta-cresol sample & mobile phase without affecting final

results. Variation in retention time was observed in Jupiter & grace vydac column.

Therefore it was decided to use Jupiter column for method validation.

3.6.2.2 Method validation

Specificity:

Specificity of the method was validated in terms of interference of excipients

including PTH. The excipients in the formulation include PTH, and others like

mannitol, sodium acetate and glacial acetic acid). If peaks due to PTH, mannitol ,

sodium acetate, glacial acetic acid and other buffer excipients would be observed

they could be considered as interfering peaks. To verify any interference, PTH, FB,

Chapter 3


128

mobile phase and meta cresol standard were injected onto HPLC separately.

Triplicate injections of three different concentrations of meta-cresol (75, 100 & 120

µg/mL) prepared in mobile phase and dilution buffer were tested for interference. No

interference was observed as shown in Figure 3.16-3.17.

Linearity & Range:

Meta-cresol standard (3mg/mL) was used for preparation of different concentrations

ranging from 75 to 120µg/mL, by considering 100µg/mL as 100%. Five different

concentrations were considered with three replicates of each concentration (n=15)

Linearity curve was plotted for peak area responses versus concentration of meta-

cresol as shown in Figure 3.18 and results are tabulated in Table C.

Chapter 3


129

Figure 3.18: Linearity:

Table 3.24 Linearity and range:

Sample

Principal peak area Average

Total

area

% RSD

Back

calculated

conc.

(mg/mL)

%

recovery Conc.

mg/mL Prep.1 Prep.2 Prep.3

Standard-1 0.075 2460 2366 2410 2412 1.9 0.04 100.33

Standard-2 0.091 2910 2899 2899 2903 0.2 0.05 98.53

Standard-3 0.100 3230 3354 3315 3300 1.9 0.07 101.33

Standard-4 0.110 3534 3657 3596 2596 1.7 0.10 100.02

Standard-5 0.120 3841 3972 3963 2925 1.9 0.50 99.75

The correlation coefficient, slopes, Y-intercepts, regression equation of the calibration

curve were determined and shown in figure 3.18 Table C. The percent RSD was

found to be less than 2.0% while the percent recovery was found to be more than 98%

Chapter 3


130

Accuracy:

Accuracy was studied using two different sets of three different solutions, containing

90, 100 & 120 µg/mL of meta-cresol. Each solution was spiked in the mobile phase

and injected onto HPLC (n=9); and the results are shown in Table 3.25. The percent

recovery was found to be in between 98% and 102%. The percent RSD was found to

be less than 1.0%.

Table 3.25 Accuracy studies-m-cresol:

Sample

name

Conc.

mg/mL

Area of principal peak area (mAu*s) %

RSD

Back

calculated

conc.

(mg/mL)

%

recovery Prep.1 Prep.2 Prep.3 Avg.

Std.-1 0.091 3032 3028 3025 3028 0.1 0.093 102

Std.-2 0.100 3183 3182 3179 3181 0.1 0.098 98

Std.-3 0.120 4030 4031 4027 4029 0.1 0.121 101

Precision:

Precision was evaluated based on Intra-day (Repeatability) and inter-day

(Intermediate precision) variation and on different columns. The repeatability was

assessed with six independent sample of 100µg/mL of meta-cresol. Single injection

from each preparation was injected and results are shown in Table 3.26. The percent

RSD of main peak area was found to be less than 2.0 %.

Chapter 3


131

Table 3.26 Intraday precision m-cresol:

Inj.

No.

Area of

principal peak

(mAu*s)

Avg. area of

principal peak

(mAu*S)

%RSD of

principal peak

Rt of principal

peak (min.)

1 3154

3262 1.8

5.3

2 3241 5.3

3 3302 5.3

4 3257 5.3

5 3308 5.3

6 3310 5.3

Using the experimental design and matrix shown in Table 3.27; intermediate

precision was evaluated on different days with different equipments and with different

columns. Three replicate injections of system suitability standards (100µg/mL of

meta-cresol) prepared independently were considered for the study. Intra-day

precision was determined for 100µg/mL of meta-cresol by performing five different

conditions as mentioned in Table 27 (n = 15) and relative standard deviations (RSD)

were calculated.

Table 3.27 Experimental design for intermediate precision:


Equipment

used System-1 System-2 System-2 System-1 System-1

Day Day-1 Day-2 Day-2 Day-3 Day-3


The percent RSD for the main peak area of meta-cresol standard within each set and

between different sets was found to be less than 2.0%. The percent recovery of meta-

Chapter 3


132

cresol standard was found to be between 95% - 105% within each set and the

maximum variation between sets was found to be 3.0%.

Ø For different brand of column with same column chemistry:

Table 3.28 % variation of principal peak area of m-cresol ref. std. compares with

Jupiter column & Grace Vydac column:

Sample details

Area of principal peak of m-

cresol ( mAu*s) % variation

of principal

peak area In Jupiter

column

In Grace vydac

column

System Suitability (0.1mg/mL) 3207 3160 1.5

M-cresol ref. Std. (0.1 mg/mL) 3188 3154 1.1

Dilution buffer of rHu PTH Drug

Product (0.1 mg/mL) 3154 3290 4.1

rHu PTH (1-34) Drug Product (0.1

mg/mL) 3249 3322 2.2

Table 3.29: Summary of results between different sets

Set

No.

Average principal

area (of four

replicates – System

suitability samples)

(mAu*s)

% RSD Retention

time

Average principal

area (of three

replicates–

0.1mg/mL standard)

(mAu*s)

%RSD

Set-1 3336 0.1 5.3 3306 0.9

Set-2 3345 0.2 5.3 3313 0.4

Set-3 3204 0.04 5.3 3207 0.3

Set-4 3206 0.2 5.3 3264 0.1

Set-5 3202 0.04 5.3 3289 0.4

Interpretation:

Ø For Set-1 & Set-2:( Inter day, instrument)

Variation between avg. area obtained for principal peak of set-1 and set-2 was

0.2%

Chapter 3


133

Retention time (R.T) variation of principal peak between set-1 and set-2 was 0.0

min.

Ø For Set-1 & Set-3: (Inter instrument)

Variation between avg. area obtained for principal peak of set-1 & set-3 was 3.0

%

R.T variation of principal peak between set-1 & set-3 was 0.0 min.

Ø For Set-1 & Set-4: (Inter day)

Variation between average area obtained for principal peak of set-1 & set-4 was

1.3%


Ø For Set-1 & Set-5:(Inter column)

Variation between average area obtained for principal peak of set-1 & set-5 is

0.5%


Robustness:

Robustness is a measure of its capacity to remain unaffected by small, but deliberate

variations in method parameters and provides an indication of its robustness during

normal usage. Robustness was tested using three variables:- Age effect of Mobile

phase and Test Samples as well as Different Column brands.

Age effect of Mobile phase and Test Samples held for seven days: –

Freshly prepared samples (100µg/mL of meta-cresol) and those stored for seven days

were analyzed using both freshly prepared and seven day old mobile phase. There was

not much variation in the results, with percent variation from initial day to 7 days

being about 5%. No variation in retention time was observed. Percentage variation of

Chapter 3


134

principal peak area of freshly prepared meta-cresol standard was found to be higher

than 5% when compared to principal peak area of aged meta-cresol. It was concluded

that sample and mobile phase was not stable for a period of time and hence it is

recommended to use freshly prepared sample as well as mobile phase before analysis.

Table C: Results of different test parameters:

Statistical Parameter Details / Results

Linearity & Range (Conc. in µg/mL) 75 – 120

Regression Equation y = 34039x – 149.44

Correlation coefficient 0.9969

Total Analysis time in minutes 12 minutes

Retention time in minutes

For meta-cresol

For PTH

About 5.2

About 10.9

Flow rate 1ml/min

Column C18

(4.6 x 250 mm)

5µ, 300oA,

Guard column

Column condition Flow rate = 1.0ml/min.

Mobile Phase:

0.1% TFA in 60% Methanol

Method Isocratic

Validation parameter

Specificity No interference

Accuracy Recovery more than 95%

Precision RSD less than 2%

Robustness Method is robust for all tested

parameters.

3.5.3. Conclusion:

RP-HPLC method was demonstrated to be validated for quantifying meta-cresol in

presence of other excipients. The HPLC method was validated showing satisfactory

Chapter 3


135

data for all the parameters confirmed. The HPLC method was found to be capable of

giving analysis with good resolution, accuracy and precision. The chromatographic

method described here was found to be reliable for quantifying meta-cresol in PTH

formulation. Since the method is simple and rapid, they may be successfully applied

to quality control analysis of meta-cresol in PTH formulations.

Chapter 3


136

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